SUMMARY Arboviruses remain an immense public health threat, causing yearly large epidemics. The overarching aim of this research is to better understand the role of the Aedes aegypti RNA interference (RNAi) pathway in antiviral defense and innate immunity, and to generate knowledge and tools for the development of new methods to control arbovirus transmission. Population replacement of wild-type with genetically modified mosquitoes incapable of pathogen transmission is emerging as a promising complement to other disease control methods. Because Ae. aegypti transmits multiple arbovirus pathogens that are frequently sympatric, it is important for a transgenic mosquito to be resistant to multiple pathogens. The siRNA pathway-mediated antiviral defense system is known to act against a broad range of viruses. However, despite the RNAi pathway's emergence as the major pan-antiviral defense system it remains understudied in mosquitoes. Our research plan is designed to test the overarching hypothesis that the A. aegypti proteins, Dicer-2 (Dcr-2), R2D2 and Argonaute-2 (Ago-2), are key components mediating an RNA silencing response that is broadly protective against arbovirus infections. We will use of siRNA-deficient loss-of-function mutants and transgenic mosquitoes over-expressing Dcr-2, Ago-2 and R2D2 that will be generated in Aim 1. With these genetic tools we will clarify the temporal and spatial specificity of the antiviral response in Aim 2, and investigate possible inter-tissue signaling and regulation of vertical transmission in Aim 3. In Aim 4 we will address interactions between the RNAi pathway and other innate immunity defense systems. This project utilizes the complementary expertise of Drs Dimopoulos and Myles with the arbovirus infection systems, mosquito transgenesis, mosquito innate immunity and RNAi/small RNA biology. Our proposed project will also generate powerful tools for studying other aspects of the RNAi pathway in Ae. aegypti biology.